J. Gen. Appl. Microbiol., 56, 81‒87 (2010)

Short Communication

Asaia spathodeae sp. nov., an acetic acid bacterium in the α-

Jintana Kommanee,1 Somboon Tanasupawat,1,* Pattaraporn Yukphan,2 Taweesak Malimas,2 Yuki Muramatsu,3 Yasuyoshi Nakagawa,3 and Yuzo Yamada2,†

1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand 3 NITE Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation (NITE), Kisarazu 292‒0818, Japan

(Received July 23, 2009; Accepted September 14, 2009)

Key Words—acetic acid ; spathodeae sp. nov.; α-Proteobacteria; 16S rRNA gene restriction analysis

The genus Asaia was fi rst described by Yamada et 2008a, b; Yukphan et al., 2004). The type strains of the al. (2000) with a single species, Asaia bogorensis, the three species were isolated respectively from fl owers type strain of which was isolated from the fl ower of an of the crown (), (Helico- orchid tree (Bauhinia purpurea). Subsequently, Asaia nia sp.), and spider lily (Hymenocallis speciosa). siamensis, Asaia krungthepensis, and Asaia lannensis Strains classifi ed in the genus Asaia were character- were described (Katsura et al., 2001; Malimas et al., ized physiologically by no or weak oxidation of ethanol to acetic acid and by no or weak growth in the pres- ence of 0.35% acetic acid (v/v) (Yamada et al., 2000). * Address reprint requests to: Dr. Somboon Tanasupawat, Tanasupawat et al. (2009) found that the two isolates Department of Microbiology, Faculty of Pharmaceutical Sci- that were isolated from fl owers of the African tulip ences, Chulalongkorn University, 254 Phayathai Road, Wang- (Spathodea campanulata) constitute a new species in mai, Bangkok 10330, Thailand. the genus Asaia during the course of the systematic E-mail: [email protected] study of acetic acid bacteria isolated from the natural † JICA Senior Overseas Volunteer, Japan International Coop- eration Agency (JICA), Shibuya-ku, Tokyo 151‒8558, Japan; environment of Thailand. Professor Emeritus, Shizuoka University, Suruga-ku, Shizuoka In this paper, we propose the name of Asaia spatho- 422‒8529, Japan. deae sp. nov. to accommodate the two isolates as a Abbreviations: BCC, BIOTEC Culture Collection, National fi fth species of the genus Asaia. Center for Genetic Engineering and Biotechnology, National Two isolates, designated as isolate GB23-2T (= BCC Science and Technology Development Agency (NSTDA), 36458T = NBRC 105894T = PCU 307T) and isolate Pathumthani, Thailand; NBRC, NITE Biological Resource Cen- GB23-3 (= BCC 31374 = NBRC 105182 = PCU 308), ter, Department of Biotechnology, National Institute of Technol- were isolated by an enrichment culture approach us- ogy and Evaluation, Kisarazu, Japan; PCU, Pharmaceutical Sci- ences, Chulalongkorn University, Bangkok, Thailand. ing a glucose/ethanol/acetic acid medium, which was The DDBJ accession numbers for the 16S rRNA gene se- composed of 1.5% D-glucose (w/v), 0.5% ethanol (v/v), quences of isolates GB23-2T and GB23-3 are respectively 0.3% acetic acid (v/v), 0.8% peptone (w/v) and 0.5% AB511277 and AB469044. yeast extract (w/v) and which was adjusted at pH 3.5 82 KOMMANEE et al. Vol. 56

(Katsura et al., 2001; Yamada et al., 1999, 2000; Yuk- 31, 40, 48, 40, and 6% respectively to isolates GB23-2T phan et al., 2004). The isolates were maintained on and GB23-3 and the type strains of Asaia bogorensis, agar slants comprised of 2.0% D-glucose (w/v), 0.5% Asaia siamensis, Asaia krungthepensis, Asaia lannen- ethanol (v/v), 0.3% peptone (w/v), 0.3% yeast extract sis, and Acetobacter aceti (Table 1). The type strains of

(w/v), 0.7% CaCO3 (w/v), and 1.5% agar (w/v). Asaia Asaia bogorensis, Asaia siamensis, Asaia krungthepen- bogorensis BCC 12264T (= NBRC 16594T), Asaia sia- sis, and Asaia lannensis gave the DNA-DNA related- mensis BCC 12268T (= NBRC 16457T), Asaia krung- ness levels of 21‒53% to the two isolates. The data thepensis BCC 12978T (= NBRC 100057T), Asaia lan- obtained indicated that the two isolates genetically nensis BCC 15733T (= NBRC 102526T), and Acetobacter constitute a new species in the genus Asaia. aceti NBRC 14818T were used for reference strains. Gene fragments specifi c for the 16S rRNA gene-en- Chromosomal DNA was prepared after Saito and coding regions of isolates GB23-2T and GB23-3 were Miura (1963). DNA base composition was determined amplifi ed by PCR, as described previously (Tanasupa- by the method of Tamaoka and Komagata (1984). wat et al., 2004; Yamada et al., 2000; Yukphan et al., DNA-DNA hybridization was performed by use of the 2004). Two primers used for PCR amplifi cation were photobiotin-labeling method with microplate wells, as 20F (5′-GAGTTTGATCCTGGCTCAG-3′; positions 9‒ described by Ezaki et al. (1989). A single-stranded and 27 by the Escherichia coli numbering system, acces- labeled DNA was hybridized with DNAs from test sion number V00348; Brosius et al., 1981) and 1500R strains in 2× SSC and 50% formamide at 48.0°C for (5′-GTTACCTTGTTACGACTT-3′; positions 1509‒1492). 15 h. The levels of DNA-DNA relatedness (%) were de- Amplifi ed 16S rRNA genes were directly sequenced termined colorimetrically (Verlander, 1992). The color with an ABI PRISM BigDye Terminator Cycle Sequenc- intensity was measured at A450 on a model VersaMax ing Ready Reaction kit on an ABI PRISM model 310 microplate reader (Molecular Devices, Sunnyvale, CA, Genetic Analyzer (Applied Biosystems, Foster, CA, USA). USA). For the direct sequencing, the following six The DNA G+C contents of isolates GB23-2T and primers were used: 20F, 1500R, 520F (5′-CAGCAGC GB23-3 were respectively 59.7 and 59.8 mol% with the CGCGGTAATAC-3′; positions 519‒536), 520R (5′-GT range of 0.1 mol% (Table 1). The data obtained were ATTACCGCGGCTGCTG-3′; positions 536‒519), 920F closely related to those of the type strains of Asaia bo- (5′-AAACTCAAATGAATTGACGG‒3′; positions 907‒ gorensis, Asaia siamensis, Asaia krungthepensis, and 926), and 920R (5′-CCGTCAATTCATTTGAGTTT-3′; Asaia lannensis (Katsura et al., 2001; Malimas et al., positions 926‒907). Multiple sequence alignments 2008a; Yamada et al., 2000; Yukphan et al., 2004). were performed for ca. 1,378 bases with the program When single-stranded and labeled DNAs from iso- CLUSTAL X (version 1.83, Thompson et al., 1997). lates GB23-2T and GB23-3 were hybridized with DNAs Gaps and ambiguous bases were eliminated from cal- from tested strains, the levels of DNA-DNA relatedness culation. Distance matrices for the aligned sequences were 100, 100, 27, 38, 43, 36, and 5% and 100, 100, were calculated by the two-parameter method of

Table 1. DNA base composition and DNA-DNA relatedness of Asaia spathodeae isolates GB23-2T and GB23-3.

DNA G+C DNA-DNA relatedness (%) of Labeled DNA from (mol%) 1234567

A. spathodeae isolate GB23-2T 59.7 100 100 27 38 43 36 5 A. spathodeae isolate GB23-3 59.8 100 100 31 40 48 40 6 A. bogorensis BCC 12264T 60.2a 21 28 100 56 59 46 8 A. krungthepensis BCC 12978T 60.3a 35 43 27 100 39 23 5 A. siamensis BCC 12268T 59.3a 41 53 22 38 100 34 6 A. lannensis BCC 15733T 60.8a 31 41 30 36 23 100 5

a Cited from Katsura et al. (2001), Malimas et al. (2008a), Yamada et al. (2000), and Yukphan et al. (2004). Abbreviations: 1, Asaia spathodeae isolate GB23-2T; 2, Asaia spathodeae isolate GB23-3; 3, Asaia bogorensis BCC 12264T; 4, Asaia krungthepensis BCC 12978T; 5, Asaia siamensis BCC 12268T; 6, Asaia lannensis BCC 15733T; 7, Acetobacter aceti NBRC 14818T. 2010 Asaia spathodeae sp. nov. 83

Kimura (1980). The phylogenetic trees based on 16S in Fig. 2, isolates GB23-2T and GB23-3 were complete- rRNA gene sequences were constructed by the neigh- ly distinguished by showing the Asp type of restriction bor-joining method (Saitou and Nei, 1987), the maxi- patterns in HpyAV digestion in spite of giving restric- mum-parsimony method (Felsenstein, 1983), and the tion patterns common to the type strains of some of maximum-likelihood method (Felsenstein, 1981) with the Asaia species examined in StyI, BsoJI, SnaBI, and the program MEGA (version 4.0; Tamura et al., 2007). HpaII digestions. In contrast, the type strains of the The confi dence values of individual branches in the four Asaia species represented the Ab type of restric- phylogenetic trees were determined by using the boot- tion patterns in HpyAV digestion. strap analysis of Felsenstein (1985) based on 1,000 The cellular fatty acid composition of isolates GB23- replications. Pair-wise 16S rRNA gene sequence simi- 2T and GB23-3 as well as the type strains of the four larities were calculated for 1,414 bases between phy- Asaia species was determined in cells grown on NBRC logenetically related strains. 804 agar at 30°C for 24 h. Methyl esters of cellular fatty The phylogenetic trees based on 16S rRNA gene acids were prepared and identifi ed according to the sequences, which were constructed by the above- instructions of the Microbial Identifi cation system mentioned three methods, showed that isolates GB23- (MIDI, Hewlett Packard, Palo Alto, CA, USA). The iso- T 2 and GB23-3 were located within the lineage of the lates contained unsaturated fatty acid of C18:1ω7c genus Asaia and formed an independent cluster from (67.5‒67.8%) and straight-chain fatty acid of C16:0 the type strains of the four known Asaia species (Fig. (9.1‒10.3%) as major components. The cellular fatty 1). However, the two isolates were related phyloge- acids profi les determined were similar to those of the netically to the type strain of Asaia siamensis with the type strains of the Asaia species (Table 2). bootstrap values respectively of 79, 77, and 83%. The Isolates GB23-2T and GB23-3 were examined for calculated pair-wise sequence similarities of isolate morphological, physiological, biochemical, and che- GB23-2T were 99.7, 99.9, 99.4, and 99.3% respectively motaxonomic characteristics (Asai et al., 1964; Hucker to the type strains of Asaia bogorensis, Asaia siamen- and Conn, 1923; Katsura et al., 2001; Tamaoka et al., sis, Asaia krungthepensis, and Asaia lannensis. The 1983; Yamada et al., 1969, 1976, 2000; Yukphan et al., similarity between the two isolates was 100%. The data 2004). The phenotypic characterization was carried obtained suggested that isolates GB23-2T and GB23-3 out by incubating the isolates and the test strains at constitute a new taxon within the genus Asaia. 30°C for 2 days on glucose/glycerol/peptone/yeast ex- To discriminate isolates GB23-2T and GB23-3 from tract agar or broth, which was composed of 1.0% D- the type strains of the four known Asaia species, a 16S glucose, 1.0% glycerol, 0.5% yeast extract and 1.0% rRNA gene restriction analysis was theoretically made peptone with or without 1.5% agar (all by w/v), unless by computer analysis using the program NEBcutter otherwise mentioned. (version 2.0, New England BioLabs, Beverley, MA, Antibiotic susceptibility of isolates GB23-2T and USA) (Yukphan et al., 2006). In the computerized cal- GB23-3 and the type strains of the four Asaia species culations, the two isolates produced restriction frag- was determined according to the conventional Kirby- ments comprised of: i) 790, 327, 214, and 83 bp in StyI Bauer method (Bauer et al., 1966). The two isolates digestion; ii) 338, 205, 172, 148, 123, 91, 87, 87, 55, and the type strains of the four Asaia species were sus- 29, 28, 24, 16, and 11 bp in BsaJI digestion; iii) no re- ceptible to nalidixic acid (30 μg), novobiocin (5 μg), striction fragment in SnaBI digestion; iv) 445, 421, 216, neomycin (30 μg), kanamycin (30 μg), gentamicin 210, 58, 53, 15, and 11 bp in HpaII digestion; v) 454, (10 μg), rifampicin (30 μg), tetracycline (30 μg), and 350, 190, 196, 169, 152, and 99 bp in HpyAV digestion. streptomycin (10 μg) but resistant to erythromycin On the other hand, the type strains of the four Asaia (15 μg), amoxycylin (10 μg), vancomycin (30 μg), am- species produced almost identical restriction frag- picillin (10 μg), bacitracin (10 units), and chloram- ments comprised of 454, 295, 190, 166, 151, 99, and phenicol (30 μg). 55 bp in HpyAV digestion. The phenotypic and chemotaxonomic characteris- The 16S rRNA gene PCR products of the two iso- tics determined are given in the species description of lates and the type strains of the four Asaia species Asaia spathodeae sp. nov. were prepared and digested respectively with the fi ve Isolates GB23-2T and GB23-3 were clearly distin- restriction endonucleases mentioned above. As shown guished from the type strains of the four known Asaia 84 KOMMANEE et al. Vol. 56

Fig. 1. Phylogenetic relationships of Asaia spathodeae isolates GB23-2T and GB23-3 based on 16S rRNA gene sequences. The phylogenetic trees were constructed by the neighbor-joining (a), maximum- parsimony (b), and maximum-likelihood (c) methods. Numbers at nodes indicate bootstrap percentages derived from 1,000 replications. species, especially by the absence of acid production cally differentiated by HpyAV digestion. from L-rhamnose as well as by production of acid from From the results obtained above, a new species is ethanol, although the two isolates had common phe- therefore proposed in the genus Asaia with the name, notypic characteristics such as peritrichous fl agella- Asaia spathodeae sp. nov. tion, weak oxidation of acetate and lactate, and growth on 30% D-glucose (w/v) in the genus Asaia (Table 3). In Description of Asaia spathodeae sp. nov. addition, the two isolates were molecular-phylogeneti- Asaia spathodeae [spa.tho’de.ae. L. gen. spathode- 2010 Asaia spathodeae sp. nov. 85

Table 2. Fatty acid profi les of Asaia spathodeae isolates GB23-2T and GB23-3.

Fatty acid (%) 1 2 3 4 5 6 Straight-chain acid C14:0 0.5 0.6 0.6 0.5 0.6 tr C16:0 10.3 9.1 11.1 9.6 8.5 6.3 C17:0 tr tr tr tr C18:0 2.3 1.9 1.2 1.0 0.8 tr Unsaturated acid C13:1 AT 12‒13 tr 0.6 tr tr C17:1 ω6c tr tr tr tr C18:1 ω7c 67.5 67.8 67.6 66.5 70.5 77.0 C19:0 cyclo ω8c tr tr 0.7 0.5 1.7 Hydroxy acid C14:0 2-OH 3.2 2.7 3.6 4.2 3.0 3.3 C16:0 2-OH 6.6 5.4 5.8 7.0 5.9 4.4 C16:0 3-OH 2.5 1.9 2.4 3.1 2.2 2.3 C18:0 3-OH 1.0 0.9 0.8 1.2 0.8 0.5 C18:1 2-OH 3.1 7.8 3.1 2.2 4.0 0.6 Summed feature 2a 1.5 1.5 1.7 2.3 1.6 1.6

Less than 0.5% fatty acids were indicated as trace (tr). a Con- tained alde-C12:0 and/or C14:0 3-OH and/or iso I-C16:1. Abbreviations: 1, Asaia spathodeae isolate GB23-2T; 2, Asaia spathodeae isolate GB23-3; 3, Asaia siamensis BCC 12268T; 4, Asaia bogorensis BCC 12264T; 5, Asaia krungthepensis BCC 12978T; 6, Asaia lannensis BCC 15733T. Fig. 2. Restriction of 16S rRNA gene PCR products of Asaia spathodeae isolates GB23-2T and GB23-3 by digestion carbonate agar. Produces 2-keto-D-gluconate and with fi ve restriction endonucleases. 5-keto-D-gluconate, but not 2,5-diketo-D-gluconate The restriction patterns were obtained by digestion with StyI (a), BsaJI (b), SnaBI (c), HpaII (d), and HpyAV (e). Lanes: 1, from D-glucose. Produces dihydroxyacetone from Asaia bogorensis BCC 12264T; 2, Asaia siamensis BCC 12268T; glycerol, but the activity is not intense. Acid is pro- 3, Asaia krungthepensis BCC 12978T; 4, Asaia lannensis BCC duced from D-glucose, D-mannose, D-galactose, D-xy- 15733T; 5, Asaia spathodeae isolate GB23-2T; 6, Asaia spatho- lose, D-arabinose, L-arabinose, D-fructose, L-sorbose, deae isolate GB23-3; M, 50-bp DNA marker. D-mannitol (weakly positive), D-sorbitol (weakly posi- tive), D-arabitol, L-arabitol, meso-ribitol, meso-erythri- ae; N. L. fem. n. Spathodea the generic name of Afri- tol, glycerol, melibiose, and ethanol. Acid is not pro- can tulip (Spathodea campanulata), the fl ower from duced from L-rhamnose, dulcitol, sucrose (weakly which the type strain was isolated]. positive in isolate GB23-3), or raffi nose. Grows on D- Cells are Gram-negative, aerobic, and rod-shaped, glucose, D-mannose, D-galactose, D-xylose, D-arabi- measuring 0.6‒1.0×1.0‒2.5 μm when cultured on glu- nose, L-arabinose (weakly positive), L-rhamnose cose/ethanol/calcium carbonate agar. Motile with peri- (weakly positive), D-fructose, L-sorbose, D-mannitol trichous fl agella. Colonies are pink, shiny, smooth, and (weakly positive in isolate GB23-2T), D-sorbitol (weakly raised with an entire margin on glucose/ethanol/calci- positive), D-arabitol, L-arabitol, meso-ribitol, meso- um carbonate agar. Grows at pH 3.0 and 3.5 at 30°C. erythritol, glycerol, melibiose, sucrose, and ethanol Grows on 30% D-glucose (w/v). Growth is weak in the but not on dulcitol, raffi nose, maltose, or lactose. The presence of 0.35% acetic acid (v/v). Methanol is not major ubiquinone is Q-10. Major cellular fatty acids are assimilated as a sole carbon source. Oxidizes acetate C18:1ω7c and C16:0. The restriction analysis of 16S and lactate to carbon dioxide and water, but the activ- rRNA genes shows the Asp type of restriction patterns ity is not intense. Grows on glutamate agar and man- in HpyAV digestion. DNA base composition is 59.7‒ nitol agar. Acetic acid is produced on ethanol/calcium 59.8 mol% G+C with a range of 0.1 mol%. 86 KOMMANEE et al. Vol. 56

Table 3. Differential characteristics of Asaia spathodeae isolates GB23-2T and GB23-3 in the genus Asaia.

Characteristic 1 2 3456 Oxidation of acetate and lactate w wwwww Growth on 30% Glucose ++++++ 0.35% Acetic acid w wwww- L-Rhamnose w w + w w + D-Mannitol w + + + w + D-Sorbitol w w + + w + Dulcitol --w - w+ Maltose --ww-- Lactose ------Acid production from L-Rhamnose --+w++ D-Mannitol w w + + w w D-Sorbitol w w w + w w Dulcitol --w - ww Sucrose - w ---- Raffi nose --w --- Ethanol + + - w - w 16S rRNA gene restriction pattern type witha HpyAV Asp Asp Ab Ab Ab Ab DNA G+C content (mol%)b 59.7 59.8 60.2 59.3 60.3 60.8

+, positive; w, weakly positive; -, negative. a For detailed information on restriction fragments, see the references (Malimas et al., 2008a; Yukphan et al., 2006). b The data were cited from the references (Katsura et al., 2001; Malimas et al., 2008a; Yamada et al., 2000; Yukphan et al., 2004) except for the two isolates. Abbreviations: 1, Asaia spathodeae isolate GB23-2T; 2, Asaia spathodeae isolate GB23-3; 3, Asaia bogorensis BCC 12264T; 4, Asaia siamensis BCC 12268T; 5, Asaia krungthepensis BCC 12978T; 6, Asaia lannensis BCC 15733T.

disk method. Am. J. Clin. Pathol., 45, 493 496. The type strain is isolate GB23-2T (= BCC 36458T = ‒ Brosius, J., Dull, T. J., Sleeter, D. D., and Noller, H. F. (1981) NBRC 105894T = PCU 307T), which was isolated from Gene organization and primary structure of a ribosomal a fl ower of the African tulip and has the DNA G+C con- RNA operon from Escherichia coli. J. Mol. Biol., 148, 107‒ tent of 59.7 mol% and ubiquinone isoprenologs com- 127. prised of 85% Q-10, 12% Q-9, and 3% Q-8. Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization Acknowledgments in microdilution wells as an alternative to membrane fi lter hybridization in which radioisotopes are used to determine This study was supported in part by The Thailand Graduate genetic relatedness among bacterial strains. Int. J. Syst. Institute of Science and Technology, TGIST, 2007, National Sci- Bacteriol., 39, 224‒229. ence and Technology Development Agency, Thailand, and Fac- Felsenstein, J. (1981) Evolutionary trees from DNA sequences: ulty of Pharmaceutical Sciences Research Fund, 2008, Chula- A maximum likelihood approach. J. Mol. Evol., 17, 368‒ longkorn University. 376. Felsenstein, J. (1983) Parsimony in systematics: Biological and References statistical issues. Annu. Rev. Ecol. Syst., 14, 313‒333. Felsenstein, J. (1985) Confi dence limits on phylogenies: An ap- Asai, T., Iizuka, H., and Komagata, K. (1964) The fl agellation proach using the bootstrap. Evolution, 39, 783‒791. and of genera Gluconobacter and Acetobacter Hucker, G. J. and Conn, H. J. (1923) Method of gram staining. with reference to the existence of intermediate strains. J. Tech. Bull. NY St. Agric. Exp. Stn., 93, 3‒37. Gen. Appl. Microbiol., 10, 95‒126. Katsura, K., Kawasaki, H., Potacharoen, W., Saono, S., Seki, T., Bauer, A. W., Kirby, W. M. M., Sherris, J. C., and Turck, M. (1966) Yamada, Y., Uchimura, T., and Komagata, K. (2001) Asaia Antibiotic susceptibility testing by a standardized single siamensis sp. nov., an acetic acid bacterium in the 2010 Asaia spathodeae sp. nov. 87

α-Proteobacteria. Int. J. Syst. Evol. Microbiol., 51, 559‒ Tanasupawat, S., Thawai, C., Yukphan, P., Moonmangmee, D., 563. Itoh, T., Adachi, O., and Yamada, Y. (2004) Gluconobacter Kimura, M. (1980) A simple method for estimating evolutionary thailandicus sp. nov., an acetic acid bacterium in the rates of base substitutions through comparative studies of α-Proteobacteria. J. Gen. Appl. Microbiol., 50, 159‒167. nucleotide sequences. J. Mol. Evol., 16, 111‒120. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Malimas, T., Yukphan, P., Takahashi, M., Kaneyasu, M., Potacha- and Higgins, D. G. (1997) The CLUSTAL X windows inter- roen, W., Tanasupawat, S., Nakagawa, Y., Tanticharoen, M., face: Flexible strategies for multiple sequence alignment and Yamada, Y. (2008a) Asaia lannaensis sp. nov., a new aided by quality analysis tools. Nucleic Acids Res., 25, acetic acid bacterium in the . Biosci. 4876‒4882. Biotechnol. Biochem., 72, 666‒671. Verlander, C. P. (1992) Detection of horseradish peroxidase by Malimas, T., Yukphan, P., Takahashi, M., Kaneyasu, M., Potacha- colorimetry. In Nonisotopic DNA Probe Techniques, ed. by roen, W., Tanasupawat, S., Nakagawa, Y., Tanticharoen, M., Kricka, L. J., Academic Press, New York, pp. 185‒201. and Yamada, Y. (2008b) Asaia lannensis corrig. Malimas et Yamada, Y., Aida, K., and Uemura, T. (1969) Enzymatic studies al. 2008. List of new names and new combinations previ- on the oxidation of sugar and sugar alcohol. V. Ubiquinone ously effectively, but not validly, published, Validation List of acetic acid bacteria and its relation to classifi cation of no. 122. Int. J. Syst. Evol. Microbiol., 58, 1511‒1512. Gluconobacter and Acetobacter, especially of the so-called Saito, H. and Miura, K. (1963) Preparation of transforming de- intermediate strains. J. Gen. Appl. Microbiol., 15, 186‒196. oxyribonucleic acid by phenol treatment. Biochim. Bio- Yamada, Y., Hosono, R., Lisdiyanti, P., Widyastuti, Y., Saono, S., phys. Acta, 72, 619‒629. Uchimura, T., and Komagata, K. (1999) Identifi cation of Saitou, N. and Nei, M. (1987) The neighbor-joining method: A acetic acid bacteria isolated from Indonesian sources, es- new method for reconstructing phylogenetic trees. Mol. pecially of isolates classifi ed in the genus Gluconobacter. Biol. Evol., 4, 406‒425. J. Gen. Appl. Microbiol., 45, 23‒28. Tamaoka, J., Katayama-Fujimura, Y., and Kuraishi, H. (1983) Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S., Analysis of bacterial menaquinone mixtures by high-perfor- Seki, T., Uchimura, T., and Komagata, K. (2000) Asaia bo- mances liquid chromatography. J. Appl. Bacteriol., 54, 31‒ gorensis gen. nov., sp. nov., an unusual acetic acid bacte- 36. rium in the α-Proteobacteria. Int. J. Syst. Evol. Microbiol., Tamaoka, J. and Komagata, K. (1984) Determination of DNA 50, 823‒829. base composition by reversed-phase high performance Yamada, Y., Okada, Y., and Kondo, K. (1976) Isolation and char- liquid chromatography. FEMS Microbiol. Lett., 25, 125‒ acterization of “polarly fl agellated intermediate strains” in 128. acetic acid bacteria. J. Gen. Appl. Microbiol., 22, 237‒245. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Yukphan, P., Malimas, T., Takahashi, M., Kaneyasu, M., Potacha- Molecular evolutionary genetics analysis (MEGA) software roen, W., Tanasupawat, S., Nakagawa, Y., Tanticharoen, M., version 4.0. Mol. Biol. Evol., 24, 1596‒1599. and Yamada, Y. (2006) Identifi cation of strains assigned to Tanasupawat, S., Kommanee, J., Malimas, T., Yukphan, P., Na- the genus Asaia Yamada et al. 2000 based on 16S rDNA kagawa, Y., and Yamada, Y. (2009) Identifi cation of Aceto- restriction analysis. J. Gen. Appl. Microbiol., 52, 241‒247. bacter, Gluconobacter, and Asaia strains isolated in Thai- Yukphan, P., Potacharoen, W., Tanasupawat, S., Tanticharoen, land based on 16S‒23S rRNA gene internal transcribed M., and Yamada, Y. (2004) Asaia krungthepensis sp. nov., spacer restriction and 16S rRNA gene sequence analyses. an acetic acid bacterium in the α-Proteobacteria. Int. J. Microbes Environ., 24, 135‒143. Syst. Evol. Microbiol., 54, 313‒316.